To start off made sure the surface was cleaned with 1% ethanol, and the Bunsen burner must be turned on. Everything performed to isolate a phage must be done in an aseptic zone, in this case it was near the Bunsen burner. 5 ml of the soil sample was gathered near WSU campus and poured into a bioreactor which already included the enriched medium and .5 ml of the M. foliorum then sat on the tube shaker for seven days.
Once the soil sample was on the shaker for a minimum of 24 hours, 1 ml of the soil mixture was transferred into a microfuge tube. The soil mixture spun in a microcentrifuge at speed for thirty seconds. Next, with a syringe with its plunger removed, a filter was attached to the top and transferred roughly half of the centrifuge mixture into it. The plunger was reinserted into the syringe, a filter the medium into a microfuge tube. The filtered medium in the microfuge tube was used as enriched lysate.
Afterward the enriched lysate was prepared, a plaque assay was performed by adding 50 ?µl of the enriched lysate into a tube of .5 of M. foliorum and letting it incubate for ten minutes. Meanwhile the enriched lysate mix was incubated for ten minute, 20 ?µl of CaCl2 was mixed with liquid top agar. Once ten minutes had passed, add the top agar mix into the phage lysate mix and swirl. The mixture of M. foliorum, phage lysate, and a separate tub of liquid top agar, it was poured into a new l-agar plate it was then incubated at 30?°C for seven days.
With a growth of a phage on the plate, a spot test was performed in aseptic conditions. A grid was drawn on the plastic side of a new l-agar plate and labeled as a negative control. A tube of liquid top agar is added 20 ?µl of CaCl?¬?¬2 and that mixture was then mixed into a tube of M. foliorum. The mixture of CaCl2, liquid top agar, and M. foliorum was drained onto a new l-agar plate. As it becomes solid on the plate, 10 “ 25 ?µl of the soil sample was transferred onto the grids that were drawn on the bottom of the plate. The l-agar plates with the mixture topped off are places in a 30?°C incubator for a week.
As the lawn was visible, a plaque streak was performed by first choosing a plaque from the plate and touching the plaque with a sterile wooden pick that was then aseptically streaked onto a new agar plate. This process was done by using a sterile wooden stick, starting at the edge of the past streak for a three separate streak where an x was noticeable. Then a new layer of agar that was mixed with M. foliorum and CaCl2 was poured onto the plate where the phage was streaked. The streaked plate was incubated at 30?°C for a week to let the bacteriophage grow.
After the streaked plate had been incubated at 30 for seven days, the next step was to devise the phage specimen located the plaque that was circled. A clean applicator was used to transfer the most isolated phage from the plate into a bioreactor that also included .5 ml of M. foliorum and 25 mL of the enriched medium and sat on the shaker for seven days.
After the enriched culture had been on the shaker for seven days, the next step was to collect the lysate and filter-sterilize. A .22 ?µm filtered syringe disbursed the now filtered enrich medium into three separate labeled microfuge tubes. Determined the titer of the HTL by ten labeled microfuge tubes from -1 to -10. Each tube was mixed with 90 ?µm of phage buffer as well as 10 ?µm of the concentrated HTL to the -1 microfuge tube. Mixed 10 ?µm from the -1 tube into the -2 tube and continue transferring 10 ?µm from the previous tube into a new tube until reaching the -10 tube with a new tip each time transferring into a new tube. A negative control was needed, so the 11th tube was infected with M. foliorum and 10 ?µm of phage buffer and letting it sit for ten minutes. As ten minutes passed, all the plates were topped off with a mixture of liquid top agar and CaCl2 and labeled appropriately. Again, it they solidified and were moved into a 30 degree Celsius incubator for the next seven days
After the plates were incubated at thirty degree Celsius for seven days, the next step was to degrade bacterial DNA / RNA in high titer lysate. First step was to disburse one mL of high titer lysate into a microfuge tube, then with gloves on, relocate to the designated nucleus work station. Added 5 ?µl of the nucleus mix into my sample, mixed by repeating, incubated at 37 degrees Celsius for ten minutes. After ten minutes of incubation, the second step was to denature the protein capsid to excrete the phage DNA. 500 ?µl of the nuclease HTL mixture was discharged onto two separate microfuge tubes with clean up resin.
The next protocol was to isolate the phage genomic DNA by attaining two DNA columns, labeled with personal initials. Columns were attached to two three mL syringes, and 1.5 mL of the phage mixture were filtered into new microfuge tubes. Put column in a new microfuge tube by first unscrewing the column from the syringe before freeing the plunger. Afterwards, withdraw the plunger from the syringe barrel and screw back the column,
Following the protocol, the next step was to rinse the salt from the DNA by using two syringe barrels filled with two mL of 80% isopropanol. Then repeat steps of isolating phage genomic DNA to have done three isopropanol washes. Afterwards, isopropanol residue was left and to remove it the columns were placed in a microfuge tube and spun at 10,000 x for a maximum of five minutes. Then, the columns were placed on a block that had been heated to 80o
C for one minute to evaporate any excess isopropanol
Eluted the DNA by incubating the columns at room temperature with 50 ?µl of ddH2O for one minute. After incubation, the samples spun again at 10,00 x for sixty seconds. The eluted phage DNA was gathered by incorporated the products from both microfuge tubes into one final tube.
First started by mildly stirring the DNA specimen by finger vertexing and with the concentrated DNA sample calculate the amount of DNA sample. Next, made sets of restriction enzyme digest reaction by getting a tube of dH2O 100 ?µg, 10X reaction buffer 20 ?µl, 3 ?µl BamHI Enzyme, 3 ?µl EcoRI Enzyme, HindIII. Reactions were set up in the format the protocol was told as. There were four reaction tubes, three restriction enzyme digest and a controlled tube with no enzyme. To indicate the reaction, started by placing the reaction tubes in tube racks and placing them in the incubator ay 37 o and my TA removed them afterwards.
Placed the 24 ?µl of reaction inside the wells of an agarose gel that was inside of the gel electrophoresis. TA provided a portion of kb ladder, dispersed all 10 ?µl of the ladder onto the gel in the lanes. After forty minutes, relocated the gel into a ziplock bag, and into a transilluminator where the DNA could be seen.